Project Summary We have developed a new method to identify molecules with antibacterial activities that rapidly discriminates between different mechanisms of action (MOA). This approach, bacterial cytological profiling (BCP), uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely MOA of new compounds within a few hours. Linnaeus Bioscience Inc. is a start up company founded to commercialize this technology and make it accessible on a fee for service basis to the pharmaceutical industry and the scientific community. During this Phase 1 project, Linnaeus will screen a highly diverse library of more than 6,000 macrocyclic compounds originally developed as kinase inhibitors in order to identify new molecules targeting multidrug resistant (MDR) Gram negative bacteria. This collection of unique chemical scaffolds has never been screened for antibacterial compounds and our preliminary data shows that it is enriched for antibacterial molecules with specific MOAs. We will screen this collection against wild type E. coli. We will employ BCP to identify the likely MOA for all hits before prioritization, and to quickly eliminate those with nonspecific or undesired activities, such as detergent-like effects on the membrane. We will counter screen against eukaryotic cell lines for cytotoxicity to eliminate highly toxic molecules. We will use classical molecular genetic and biochemical approaches to identify the precise molecular target of the prioritized hits, and to evaluate the potential for resistance to emerge. We will identify the most chemically tractable priority hit series and generate early leads through medicinal chemistry efforts. The goal of these studies is to identify early lead molecules that can be developed further. These studies will demonstrate that BCP is a robust whole cell imaging based method capable of identifying molecules with antibacterial properties on an industrial scale. This platform will be made widely available on a fee for service basis.